Bone marrow fragments or cell suspensions of murine, rat, guinea pig, rabbit, porcine, and canine origin form osteogenic tissue when transplanted into heterotopic sites in vivo. In closed systems such as diffusion chambers, bone marrow constituents form either bone or bone and cartilage depending on the size of the chamber. In open systems such as implantation under the kidney capsule where neovascularization can occur, bone ossicies surround a hematopoietic marrow resulting in formation of a bone marrow organ (Ashton et al., Clin. Orthop., 151:294, 1980; Bab et al., J. Cell Sci., 84:139, 1986; Friedenstein, Bone Min. Res., 7:243, 1990). Bone marrow organs are characterized by the property of self-maintenance; that is, they provide physiological support to the hematopoietic tissue localized therein and remain vital for the lifetime of the recipient animal. In contrast, bone induced by transitional epithelium of urinary bladder, demineralized bone matrix, or isolated factors is not self-maintained in heterotopic sites without the continuous presence of an inducer.
MSFs become the predominant adherent cell type when human bone marrow is cultured in vitro. In cultures generated from single-cell suspensions of marrow, MSFs grow in colonies, each derived from a single precursor cell termed the colony forming unit fibroblast (CFU-F). In addition to their fibroblast-like morphology, MSFs share a variety of fibroblastic features but lack the basic characteristics of endothelial cells and macrophages. After extended culture, MSFs of mouse, rat, guinea pig, and rabbit origin have been reported to maintain the ability to form at least five types of connective tissue in transplantation systems, including bone, cartilage, fibrous tissue, adipose tissue and hematopoiesis-supporting reticular stroma (Ashton et al., Clin. Orthop., 151:294, 1980; Gerasimov et al., Bull Exper. Biol. Med., 101:802, 1986; Chailakhyan et al., Bull. Exper. Biol. Med., 86:1533, 1978; Patt et al., Exp. Hematol., 10:738, 1982; Friedenstein et al., Cell Tissue Kinet., 20:263, 1987; Kuznetsov et al., Bull. Exper. Biol. Med., 108:1186, 1989; Bennett et al., J. Cell Sci., 99:131, 1991). Thus, MSF populations contain pluripotent stromal stem cells that are capable of proliferation, renewal and differentiation into several phenotypes. These stromal stem cells give rise to lineages distinct from those of hematopoietic stem cells. More mature osteoblastic cells isolated from rodent bone such as calvariae lose the stem cell properties of MSFs and lack the ability to form bone when transplanted after long term culture. After short-term culture they form bone, but not a bone marrow organ or cartilage.
MSFs have been loaded into various delivery vehicles and implanted into immunodeficient mice to study bone formation in vivo. Krebsbach et al. (American Society for Bone and Mineral Research, Baltimore, Md., September 1995) loaded cultured mouse MSFs into Gelfoam.TM. sponges, polyvinyl sponges, or block ceramic disks prior to implantation into immunodeficient mice. Bone formation was initiated within two weeks and increased with time. Only limited bone formation, if any, was also observed when human MSFs were cultured and loaded into Gelfoam.TM. sponges. Bone formation was far more exuberant in ceramic blocks when implanted into mice.
The osteogenic potential of human bone cells has also been studied using several experimental models. Primary bone cells derived from children occasionally formed bone and cartilage, but did not support hematopoiesis after intramuscular transplantation into cortisone-pretreated mice (Yamamoto et al., J. Bone Min. Res., 6:45, 1991). When primary bone cells were transplanted within diffusion chambers, bone formation occurred only in the presence of osteogenic inducers (Davies, Cell Biol. Intern. Rep., 11:125, 1987; Gundle et al., Bone, 16:597, 1995). When viable fragments of human bone were transplanted into immunodeficient mice pretreated with radiation, osteoblasts survived and deposited new bone upon preexisting bone fragments (Boynton et al., Bone, 18:321, 1996).
In contrast to rodent marrow cells, the osteogenic potential of human bone marrow cells is less well characterized. When adult human marrow cells were transplanted in diffusion chambers, only unmineralized fibrous tissue was formed; however, marrow cells from young children occasionally developed osteogenic tissue (Bab et al., Bone Min., 4:373, 1988; Gundle et al., ibid). Like human bone cells, human MSFs showed no signs of osteogenesis in diffusion chambers transplanted intraperitoneally into nude mice (Ashton et al., Bone, 6:313, 1985; Gundle et al., ibid, Haynesworth et al., Bone, 13:81, 1992) unless they had been cultured in the presence of osteogenic inducers (Gundle et al., ibid).
There is a need for a method of stimulating new human bone formation in vivo. There is also a need for a model system which will allow the determination of compounds which inhibit or stimulate human bone formation and as a method for testing molecular engineering techniques. The present invention addresses these needs.